Team:Hawaii/Notebook/2008-09-23

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===Sequencing===
===Sequencing===
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:<strong> Margaret</strong>
:<strong> Margaret</strong>
[[Image:rep_9_23.jpg|right|thumb|300px|1st lane is ladder, 2nd lane is rep amplified from a colony, 3rd lane is rep amplified from a restriction product of rep from the same colony.]]
[[Image:rep_9_23.jpg|right|thumb|300px|1st lane is ladder, 2nd lane is rep amplified from a colony, 3rd lane is rep amplified from a restriction product of rep from the same colony.]]
:*PCR of rep, ran a gel to verify size, and exo-sap. will send in tomorrow
:*PCR of rep, ran a gel to verify size, and exo-sap. will send in tomorrow
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<div style="clear:both;">
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===PCR: pRL1383a MCS Replacement===
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:'''Norman'''
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[[Image:20080923-emergency_vector_pRLBB_vector_insert_PCR.annotated.jpg|thumb|right|making x6 Parallel MCS Repalcement fragmenets via PCR using OLD PRIMERS!!! [[Media:20080923-emergency_vector_pRLBB_vector_insert_PCR.jpg|download unannotated]]]]
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[[Image:20080924-emergency_vector_pRLBB_vector_insert_PCR_redo.annotated.jpg|thumb|right|making x6 Parallel MCS Replacement fragmenets via PCR Redo!!! with clean uncontaminated primers [[Media:20080924-emergency_vector_pRLBB_vector_insert_PCR_redo.jpg|download unannotated]]]]
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:* x6 parallel MCS replacements fragment by PCR amplification.  PCR out each MCS replacement insert with HindIII-VF2BB_fx._sb.1 and BamHI-VRBB_rx._sb.1
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:*# BBa_I52002 from pSB4A5 (Spring 2008 Plate 1022 1C)
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:*# BBa_I52001 from pSB4T5 (Spring 2008 Plate 1020 1A)
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:*# BBa_I52002 from BBa_I51020 (BBa_I51020 Base Vector from glycerol stock Inventory [http://packrat.stjohn.hawaii.edu/prestinglab/wiki/INV-Z80-R1-A3 INV-Z80-R1-A3])
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:*# BBa_P1010 from pSB1A3 (pSB1A3 from glycerol stock Inventory [http://packrat.stjohn.hawaii.edu/prestinglab/wiki/INV-Z80-R1-A3 INV-Z80-R1-A3])
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:*# BBa_P1010 from pSB1A7 (pSB1A7 from glycerol stock Inventory [http://packrat.stjohn.hawaii.edu/prestinglab/wiki/INV-Z80-R1-A3 INV-Z80-R1-A3])
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:*# BBa_J33207 from pSB1A2 ([http://partsregistry.org/wiki/index.php/Part:BBa_J33207 BBa_J33207] harboring pSB1A2 from plasmid prep)
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:* x2 (or x3) Controls
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:*# positive control: [http://partsregistry.org/wiki/index.php/Part:BBa_K125805 BBa_K125805] from pSB1A3 ([http://partsregistry.org/wiki/index.php/Part:BBa_K125805 BBa_K125805] harboring pSB1A3 from glycerol stock Inventory [http://packrat.stjohn.hawaii.edu/prestinglab/wiki/INV-Z80-R1-D4 INV-Z80-R1-D4])
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:*# negative control: H<sub>2</sub>O primer set used on 1-7
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:*# negative control: H<sub>2</sub>O old Bam-VF2BB Hind-VRBB primers
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<div style="clear:both;">
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::::Are you sure the controls had contaminated primers, that they weren't contaiminated during the pipetting process? There's only a single band in each old primer control lane (contaminants usually give more than one band). Also, the band is different between the first gel (~850bp?) and the second (~920bp).*'''[[User:Gracek|Gracek]] 07:35, 25 September 2008 (UTC)'''
= Discussion =
= Discussion =

Latest revision as of 07:36, 25 September 2008

Projects Events Resources
Sponsors Experiments Milestones Protocols
Notebook (t) Meetings (t)

Things we did today

Wetlab work

Plasmid prep

Grace
  • pSB1A3
  • rbs+GFPf+tt #1, 2
  • nir+rbs+GFP #1, 4, 7
  • nir+rbs+slr1+GFPf #2, 8
  • nir+rbs+pilA #18

Sequencing

Margaret
1st lane is ladder, 2nd lane is rep amplified from a colony, 3rd lane is rep amplified from a restriction product of rep from the same colony.
  • PCR of rep, ran a gel to verify size, and exo-sap. will send in tomorrow

PCR: pRL1383a MCS Replacement

Norman
making x6 Parallel MCS Repalcement fragmenets via PCR using OLD PRIMERS!!! download unannotated
making x6 Parallel MCS Replacement fragmenets via PCR Redo!!! with clean uncontaminated primers download unannotated
  • x6 parallel MCS replacements fragment by PCR amplification. PCR out each MCS replacement insert with HindIII-VF2BB_fx._sb.1 and BamHI-VRBB_rx._sb.1
    1. BBa_I52002 from pSB4A5 (Spring 2008 Plate 1022 1C)
    2. BBa_I52001 from pSB4T5 (Spring 2008 Plate 1020 1A)
    3. BBa_I52002 from BBa_I51020 (BBa_I51020 Base Vector from glycerol stock Inventory [http://packrat.stjohn.hawaii.edu/prestinglab/wiki/INV-Z80-R1-A3 INV-Z80-R1-A3])
    4. BBa_P1010 from pSB1A3 (pSB1A3 from glycerol stock Inventory [http://packrat.stjohn.hawaii.edu/prestinglab/wiki/INV-Z80-R1-A3 INV-Z80-R1-A3])
    5. BBa_P1010 from pSB1A7 (pSB1A7 from glycerol stock Inventory [http://packrat.stjohn.hawaii.edu/prestinglab/wiki/INV-Z80-R1-A3 INV-Z80-R1-A3])
    6. BBa_J33207 from pSB1A2 ([http://partsregistry.org/wiki/index.php/Part:BBa_J33207 BBa_J33207] harboring pSB1A2 from plasmid prep)
  • x2 (or x3) Controls
    1. positive control: [http://partsregistry.org/wiki/index.php/Part:BBa_K125805 BBa_K125805] from pSB1A3 ([http://partsregistry.org/wiki/index.php/Part:BBa_K125805 BBa_K125805] harboring pSB1A3 from glycerol stock Inventory [http://packrat.stjohn.hawaii.edu/prestinglab/wiki/INV-Z80-R1-D4 INV-Z80-R1-D4])
    2. negative control: H2O primer set used on 1-7
    3. negative control: H2O old Bam-VF2BB Hind-VRBB primers
Are you sure the controls had contaminated primers, that they weren't contaiminated during the pipetting process? There's only a single band in each old primer control lane (contaminants usually give more than one band). Also, the band is different between the first gel (~850bp?) and the second (~920bp).*Gracek 07:35, 25 September 2008 (UTC)

Discussion

Quote of the Day

History is the only laboratory we have in which to test the consequences of thought. - Étienne Gilson


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